ABSTRACT (CETR RP5, Sztein, PL) Infection with Salmonella spp. due to ingestion of contaminated food and water, including typhoidal (caused largely by S. Typhi (ST) and S. Paratyphi A (PA)), as well as non-typhoidal (NTS) and invasive NTS (iNTS) infections are major public health concerns in many areas of the World, including in the U.S. where NTS causes 1.2 million illnesses annually. The rapid increase in multidrug resistance (MDR) and the lack of vaccines against PA, NTS or iNTS have added a new sense of urgency for the development of vaccines against these pathogens, and ideally broad-spectrum vaccines. One of the major obstacles in developing vaccines against Salmonella spp. is that the precise immunological correlates of protection (CoP) against either infection with wild-type (wt) organisms or vaccines remain unknown, in part because ST and PA are human-restricted infections. The use of specimens from volunteers vaccinated and/or challenged with wt ST has begun to uncover immunological T cell-mediated immunity (T-CMI) effector mechanisms which might be associated with clinical outcome following challenge. No similar data is available for PA since the first wt PA challenge has just been performed. A critical gap also remains in our understanding of the mechanisms of antigen presentation for different Salmonella spp., which may underlie the development of effective adaptive T- CMI and B cell responses, as well as the immune responses elicited in the gut microenvironment following exposure to typhoidal and NTS infections. For these reasons, the overall goal of this application is to advance the development of vaccines against PA as well as broad-spectrum vaccines against enteric fevers, iNTS, and NTS, by identifying protective cross- reactive Salmonella spp. humoral, T effector, and regulatory immune responses, both systemically and in the gut microenvironment, and by defining the mechanisms of antigen presentation against ST, PA, iNTS and NTS that impact adaptive immune cell programming. To achieve these goals we will use PBMC from (i) a challenge study with wt PA with known clinical outcomes (e.g., non-disease or disease), (ii) a study with attenuated PA vaccine candidate strain CVD 1902 followed by challenge with wt PA, (iii) in vitro T cell priming systems and (iv) three human intestinal models: (a) bioengineered 3-D organoids, (b) enteroids, and (c) explants to perform the following Aims: Aim 1. Evaluate whether a defined set of B and T cellular responses in circulation are associated with protection from bacteremic infection and/or bacteremia-negative clinical disease (e.g., fever) following an oral challenge with wt PA in humans, and which regulatory mechanisms are involved in generating these responses. Aim 2. Evaluate whether interactions between ST, PA, iNTS and NTS and dendritic cells (DC) lead to the activation of diverse pathways which in turn determine the priming of defined T cell responses. Aim 3. Contrast molecular biomarker changes and function elicited in gut innate immune cells by various Salmonella serovars and the corresponding isogenic vaccine strains.